Sections (2 μm) from formalin-fixed, paraffin-embedded tumor nodules were processed with DeadEnd Fluorometric TUNEL (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The sections were examined using a light microscope (BX53; Olympus, Tokyo, Japan). Image analysis was performed using open source ImageJ software (http://rsb.info.nih.gov/ij/ ).
Deadend fluorometric tunel
Manufactured by Promega
Sourced in United States
The DeadEnd Fluorometric TUNEL System is a lab equipment product designed to detect and quantify apoptosis, a type of programmed cell death. The core function of this product is to label the fragmented DNA of apoptotic cells, allowing for their identification and analysis using fluorescence-based detection methods.
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Lab products found in correlation
Zen software, by Zeiss (2 mentions)
Axio imager z1 microscope, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Phospho gskα β, by Cell Signaling Technology (1 mentions)
Rank ligand rankl, by R&D Systems (1 mentions)
1 25 dihydroxyvitamin d3, by Merck Group (1 mentions)
Phospho ikkα β, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Promega (1 mentions)
Tartrate resistant acid phosphatase staining kit, by Merck Group (1 mentions)
Phospho akt thr308, by Cell Signaling Technology (1 mentions)
Prostaglandin e2, by Merck Group (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
α mem, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ly294002, by Merck Group (1 mentions)
M csf, by R&D Systems (1 mentions)
Collagen gel, by Nitta Gelatin (1 mentions)
14 protocols using deadend fluorometric tunel
1
TUNEL Assay for Apoptosis Analysis
2
Osteoclastogenesis Signaling Pathway Assay
3
Immunohistochemical Analysis of Aortic Tissue
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Human aortic samples were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm intervals. Immunohistochemical staining was performed using established methods [17 (link)]. To remove the paraffin, sections were treated with xylene and rehydrated. Then, they were incubated with 3% H2O2 for 10 min at room temperature and washed 3 times with phosphate-buffered saline (PBS). After blocking with serum for 30 min, the sections were incubated with primary antibodies against YAP (1 : 1000 dilution, Cell Signaling), α- smooth muscle actin (α-SMA, 1 : 500 dilution, Sigma), Bcl-2 (1 : 1000 dilution, Cell Signaling), and cleaved caspase-3 (1 : 300 dilution, Cell Signaling), followed by incubation with the ChemMate EnVision System (Dako). ImageProPlus 3.0 (ECIPSE80i/90i) was used to capture the images and analyze the results. For cryostat sections, human and mouse aortic samples were fixed in 4% paraformaldehyde, embedded in optimum cutting temperature (OCT) compound, frozen in liquid nitrogen, and sectioned at 5 μm intervals. DeadEnd Fluorometric TUNEL (Promega) was used to detect the apoptotic cells. Apoptotic VSMCs were detected by TUNEL and α-SMA double staining before confocal fluorescence microscopy analysis (Leica Microsystems).
4
Apoptosis Quantification via TUNEL
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Percentage of apoptotic cells was determined using DeadEnd™ Fluorometric TUNEL (TdT-mediated dUTP Nick-End Labeling) System (Promega; # G3250) following the manufacturer instructions.
5
Chitosan Nanoparticles for Targeted Gene Delivery
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Chitosan (25 kDa) was purchased from Coyotefoods Biopolymer and Biotechnology Mexico. Cell-culture media, fetal bovine serum, and cell culture supplements were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Tripolyphosphate (TPP) was purchased from Sigma-Aldrich (St Louis, MO, USA). MNPs were obtained from OZ Biosciences (CombiMag; Marseille, France). In vivo DogtorMag™ transfection reagent designed for in vivo targeted transfection was also provided by OZ Biosciences. A plasmid purification kit was purchased from Thermo Fisher Scientific. A caspase 3 (active) red-staining kit was purchased from CTR Scientific (Monterrey, Mexico). A Wizard® Genomic DNA Purification kit, EcoR1, XbaI, and HindIII restriction enzymes, and DeadEnd fluorometric TUNEL (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling) system were purchased from Promega Corporation (Fitchburg, WI, USA).
6
Immunofluorescence Staining of Paraffinized Tissues
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Paraffinized sections (mouse and human) were prepared for immunofluorescence staining by heating the slides for 15 min at 55°C in an oven, deparaffinized (2 x 100% xylene 5 min each, 2 x 100% ethanol 5 min each, 2 x 95% ethanol 5 min each, 70% ethanol for 5 min) and rinsed in dH2O for 5 min. Antigen retrieval was performed by heating the slides at 95°C for 20 min in HistoVT pH 7.0 (Nacalai USA) for all antibodies used. Specimens were blocked in 5% goat serum PBS-T for 15 min at RT before incubating with primary antibody diluted in 1% goat serum PBS-T overnight at 4°C. For primary antibodies produced in goat, donkey serum was used as the blocking agent. Each slide was rinsed three times in PBS, for 5 min each. Specimens were incubated in fluorochrome-conjugated secondary antibody diluted in 1% goat serum PBS-T for 1 hr at RT in the dark. After rinsing as above, VectorShield with DAPI and coverslip were mounted and slides were allowed to cure overnight at 4°C in the dark before image acquisition. For apoptosis assessment, we used DeadEnd Fluorometric TUNEL (Promega) and performed the staining as recommended by manufacturer. A list of antibodies used can be found in Key Resources Table .
Images were acquired using either ApoTome.2 (Zeiss) without structured illumination or LSM780 (Zeiss) and analyzed using ImageJ software unless otherwise stated.
Images were acquired using either ApoTome.2 (Zeiss) without structured illumination or LSM780 (Zeiss) and analyzed using ImageJ software unless otherwise stated.
7
TUNEL Assay for Apoptosis Quantification
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TUNEL (DeadEnd Fluorometric TUNEL, Promega, Madison, WI, USA) was used to label apoptotic cells in one-month-old animals, following our previously published protocol [10 (link),14 (link)]. The sections were analyzed on an Axio Imager Z1 microscope (Carl Zeiss, Jena, Germany) using Zeiss Zen software (Carl Zeiss, Jena, Germany). Eyes of eleven animals (VPP: n = 11, control: n = 11) were included in the morphometric analyses and their TUNEL-positive cells were counted and normalized to the area of the ONL [mm2]. There was no sex-specific difference in the number of TUNEL positive cells between VPP (male (n = 6) versus female (n = 5)) animals.
8
Detecting Apoptotic Glomerular Cells
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Apoptotic glomerular cells in GN were detected by a DeadEnd fluorometric TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) system following the manufacturer’s instructions (Promega, Madison, WI). Fragmented DNA was identified by incorporating fluorescein-12-dUTP at the 3′-OH DNA ends with terminal deoxynucleotidyl transferase in terminal deoxynucleotidyl transferase incubation buffer. The nuclei of the cells were counterstained with propidium iodide. Apoptotic glomerular cells were counted in 30 full-size glomeruli.
9
Detecting Vascular Cell Apoptosis via TUNEL
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The DeadEnd Fluorometric TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) System (G3250, Promega, Madison, WI) was used to detect the vascular cell apoptosis according to the manufacturer’s instructions. In belief, the aortic tissue was sectioned (7-μm-thick, ≈1 mm apart), and 8 serial sections for each infrarenal abdominal aorta were incubated with equilibration buffer for 10 minutes, then TdT (terminal deoxynucleotidyl transferase) reaction mixture was added to the tissues for 1-hour incubation at 37°C followed by 2× SSC buffer used for stopping the reaction. The data were obtained using a confocal microscope (Leica, Wetzlar, Germany) and evaluated blindly by 2 independent investigators.
10
Cell Viability and Apoptosis Assays
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Culture ware and
other plastic consumables were purchased from Nunc, Denmark. Cancer
cell lines were purchased from American Type Cell Culture, ATCC, USA.
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
reagent was obtained from thermofisher scientific, USA, ApoAlert Annexin
V, Clontech, USA, DeadEnd Fluorometric TUNEL Promega, USA. All required
chemicals and cell culture reagents were obtained from Sigma-Aldrich,
USA.
other plastic consumables were purchased from Nunc, Denmark. Cancer
cell lines were purchased from American Type Cell Culture, ATCC, USA.
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
reagent was obtained from thermofisher scientific, USA, ApoAlert Annexin
V, Clontech, USA, DeadEnd Fluorometric TUNEL Promega, USA. All required
chemicals and cell culture reagents were obtained from Sigma-Aldrich,
USA.
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